Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of the blastocyst stage embryo that can differentiate in vivo and in vitro into all cell lineages of the adult animal. The ability of these cells to adopt.multiple fates makes ES cells prime candidates for use in stem cell therapies. The mechanisms that maintain sternness in human ES cells (hESCs) are poorly understood. The first part of the proposed studies will examine the signaling pathways responsible both for maintenance of sternness and for neural differentiation of hESCs. Successful use of hESCs in transplantation strategies will require an extracellular milieu appropriate for directing survival and differentiation of the cells. We have found that a self-assembling peptide amphiphile that can be safely injected directly into neural tissue promotes neuronal differentiation of neural stem cells and facilitates functional recovery after spinal cord injury. The second aspect of the proposed research will examine the effects of such nanoengineered materials on hESCs and will define the mechanisms underlying effects of the nanogels on hESC properties. The stem cell technology and nanotechnology that are developed as part of these studies will then be used to address the problem of spinal cord injury. The lack of regenerationfollowing injury to the adult spinal cord reflects a number of factors including the presence of molecules inhibitory to axon outgrowth, the paucity of molecules that foster regeneration, and the formation of a cavity and of a glia scar that can physically impede axonal outgrowth. This study will therefore adopt a multifactorial approach that addresses each of these issues. First, hESCs will be engineered to secrete blockers of the major, well-studied inhibitors, Nogo, MAG, and Omgp. Second, hESCs will be engineered to secrete neurotrophic factors that have been shown to facilitate motor and sensory tract regeneration. Third, the nanoengineered self-assembling gel will be used in conjunction with the hESCs to fill the cavity and to inhibit formation of the glia scar. The long term goal of these studies is to develop techniques for combining advances in stem cell biology and nanotechnology for the repair/ regeneration of the damaged central nervous system. The studies described in this proposal will only utilize NIH approved embryonic stem cell lines lines including WA01, UC06, and TE03.